- Email: [email protected]
Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice
Biochemical and Biophysical Research Communications 412 (2011) 752–756
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice Rachel Young a,⇑, Sandrine Le Guillou a, Gaëlle Tilly a, Bruno Passet a, Marthe Vilotte a, Johan Castille a, Vincent Beringue b, Fabienne Le Provost a, Hubert Laude b, Jean-Luc Vilotte a a b
INRA UMR 1313, Equipe Différenciation et Spécialisation Cellulaires (DISC), Génétique Animale et Biologie Intégrative (GABI), 78350 Jouy-en-Josas, France INRA UR892, Maladies à Prion/Macroassemblage Protéiques, Virologie Immunologie Moléculaires, 78350 Jouy-en-Josas, France
a r t i c l e
i n f o
Article history: Received 8 August 2011 Available online 17 August 2011 Keywords: Transgenic reporter mice Shadoo Paralogue of prion protein Gonadic expression LacZ Fertility and reproduction
a b s t r a c t The protein Shadoo (Sho) is a paralogue of prion protein, and encoded by the gene Sprn. Like prion protein it is primarily expressed in central nervous system, and has been shown to have a similar expression pattern in certain regions of the brain. We have generated reporter mice carrying a transgene encompassing the Sprn promoter, exon 1, intron 1 and the 50 -end of exon 2 driving expression of either the LacZ or GFP reporter gene to study the expression profile of Shadoo in mice. Expression of the reporter genes was analysed in brains of these transgenic mice and was shown to mimic that of the endogenous gene expression, previously described by Watts et al. [1]. Consequently, the Sprn-LacZ mice were used to study the spatial expression of Sho in other tissues of the adult mouse. Several tissues were collected and stained for b-gal activity, including the thymus, heart, lung, liver, kidney, spleen, intestine, muscle, and gonads. From this array of tissues, the transgene was consistently expressed only in specific cell types of the testicle and ovary, suggesting a role for Shadoo in fertility and reproduction. These mice may serve as a useful tool in deciphering the regulation of the prion-like gene Sprn and thus, indirectly, of the Shadoo protein. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The prion protein family consists of three proteins; Prion, Doppel, and Shadoo. Expression profiles of both Prion and Doppel have been characterized in several species including mouse, human, goat, and sheep, however little has been reported on the expression of Sprn. Prion (PrPc) is a ubiquitously expressed protein, with a high expression level in the brain. The function of this GPI-anchored protein is not yet clear, as Prnp-knockout mice [2,3], cattle [4] and goat [5] suffer from no drastic phenotype. An abnormal form of the prion protein, PrPSc, is the infectious agent in prion diseases such as Bovine Spongiform Encephalopathy, Creutzfeldt–Jakob Disease, Scrapie and Chronic Wasting Disease. Various roles have been proposed for PrP in neuroprotection, cellular homeostasis, response to oxidative stress, cell proliferation and differentiation, synaptic function and signal transduction [6–9]. Doppel is highly expressed in the testis, and to a lesser extent the ovaries and spleen. Knockout of the gene encoding Doppel (Prnd) resulted in male sterility [10,11], both studies performed resulted in phenotypes suggesting a role for Doppel in spermatogenesis and/or male fertility. A role for Doppel in early sex differ-
⇑ Corresponding author. Fax: +33 1 34 65 24 78. E-mail address: [email protected] (R. Young). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.049
entiation in goats was also suggested, due to its expression pattern in testis and ovary at various developmental stages [12]. Shadoo expression has been less well characterized than that of Doppel and PrP. The Sprn gene was identified in 2003 by Premzl et al. [13]. Since then homology shared between the N-terminal of the Prion and Shadoo protein structures [14], and overlapping expression pattern in the brain [1] has raised the question of its ability to compensate for lack of PrPc in Prnp-knockout animals [15]. Here we describe transgenic reporter mice generated using Sprn-regulatory sequences and either a green fluorescent protein (GFP) or LacZ reporter gene. These mice were used to analyse the expression profile of Sprn in the adult mouse. 2. Materials and methods All animal manipulations were done according to the ‘‘French Commission de Génie Génétique’’ recommendations. 2.1. Generation of Sprn-GFP and Sprn-LacZ transgenic mice A 5.3 kb genomic fragment including exon 1, intron 1 and the 50 -end of exon 2 of the mouse Sprn gene was amplified using the following primers: sense 50 -CAAGAGGATCTCTATAAACTCAAGGCTA-30 and antisense 50 -AATCTGCGAGAAGAGGGTGGAA-30 . It was
R. Young et al. / Biochemical and Biophysical Research Communications 412 (2011) 752–756
753
Table 1 Sequences of primers used for genotyping and RT-PCR analysis. Gene
Forward primer
Sprn-GFP Sprn-LacZ Endogenous Sprn Beta-actin
Reverse primer
0
0
5 -ACATGAAGCAGCACGACTTCTT-3 50 -GGTCCTAAACCACGCTCCAC-30 50 -CAGTCGTGAGCTCTGCCTAA-30 50 -CACCAGTCCGCCATGGATG-30
50 -TTCTGCTTGTCGGCCATGATATAGACGTT-30 50 -GTTTTCCCAGTCACGACGTTG-30 50 -GGAACAGCTGTCACAGAGGA-30 50 -TCCCCACCATCACACCCTG-30
A
B
GFP
LacZ
Number of mice born
297
208
% Transgenic
35% (6)
33% (3)
% Lines expressing
100% (6/6)
100% (3/3)
C
Fig. 1. Transgene structure, transmission and expression. (A) Two transgenes were made to report on Sprn gene expression in mice, one using a GFP reporter gene and the other using LacZ. In both cases 5.3 kb of the regulatory region of the Sprn gene was used. (B) The table above summarises the mouse lines established for both transgenes. (C) Brain expression profile of Sprn-GFP (green) and Sprn-LacZ (blue). Both transgenes gave the same profile of expression. Cells positive for the transgene resemble the Purkinje cells of the cerebellum and the neurons of the thalamus. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
inserted into either pEGFP-N1 vector or pCMVb vector, to make the Sprn-GFP and Sprn-LacZ constructs respectively. Recombinant vectors were digested with either SalI-AflII (SprnGFP) or SalI (Sprn-LacZ), gel-purified and micro-injected into FVB/N Prnp0/0 oocytes [2,16]. Offspring were genotyped by PCR using the following primers: GFP sense 50 -ACATGAAGCAGCACGACTTCTT-30 , GFP antisense 50 -TTCTGCTTGTCGGCCATGATATAGACGTT-30 , or La cZ sense 50 -GGTCCTAAACCACGCTCCAC-30 , LacZ antisense 50 -GTTT TCCCAGTCACGACGTTG-30 . 2.2. Analysis of transgene expression Three male and three female transgenic mice from each line (six GFP and three LacZ lines) were sacrificed by cervical dislocation.
Three male and three female wild type mice were also used as negative controls. Tissues were collected for either cryosections or RNA extraction in PBS or liquid nitrogen respectively. Sprn-LacZ tissues were fixed in LacZ fix (0.2% gluteraldehyde, 5 mM EGTA, 100 mM MgCl2, PBS) for 4 h then incubated overnight in 20% sucrose solution. Sprn-GFP tissues were incubated in 20% sucrose overnight without fixation. Tissues were mounted in Cryomount (Histolabs) at 25 °C in a cryostat, and then stored at 80 °C. Ten micron cryosections of Sprn-GFP or Sprn-LacZ tissues were cut and collected on Superfrost slides (Thermo Scientific). Sprn-GFP sections were counterstained with DAPI using Vectorshield + DAPI (Vectorlabs). Sprn-LacZ sections were re-fixed for 10 min in LacZ fix, then washed in a detergent wash (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Igepal, PBS) 2 5 min, then
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R. Young et al. / Biochemical and Biophysical Research Communications 412 (2011) 752–756
Fig. 2. Expression of Sprn-LacZ. (A) Brain expression of Sprn-LacZ. Expressing regions include the dentate gyrus, hippocampus, cerebellum and thalamus. The expression pattern given by the transgene mirrors that of the endogenous gene (published by Watts et al. [1]). (B) Sprn-LacZ tissue-distribution. Other tissues were tested, including; testicle, ovary, liver, kidney, lung, thymus, muscle, heart, mammary gland, and intestine. The transgene was only expressed in brain, testicle and ovary. In the testicle, SprnLacZ was expressed in the leydig cells. In the ovary Sprn-LacZ was expressed in the granulosa cells of the developing follicle. Non-transgenic mice were used as negative controls for (A) and (B), these showed no staining (data not shown). (C) RT-PCR analysis of endogenous Sprn correlates with expression of the Sprn-LacZ transgene. Abbreviations: BR, brain; SPL, spleen; TES, testis; INT, intestine; KID, kidney; LV, liver; OV, ovary; THY, thymus; HRT, heart.
stained overnight at 37 °C in a humid staining chamber with LacZ stain (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Igepal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 20 mM Tris buffer pH 7.3, 1 mg/ml X-gal, PBS) protected from the light. The sections are then washed 3 5 min in PBS then rinsed in dH2O, air dried and mounted with Permount (Fischer Scientific). Sprn-GFP sections were analysed by fluorescent light microscopy using a Leica microscope. Sprn-LacZ sections were analysed by light microscopy on the same microscope. 2.3. RNA extractions and RT-PCR RNA collected for RT-PCR analysis was extracted using the RNeasy Lipid Tissue Mini kit (Qiagen). RNA concentration was calculated by electro-spectrophotometry, then 5 lg of each sample was reverse transcribed using the Superscript III Reverse Transcription kit (Invitrogen).
RT-PCR analysis was performed for both endogenous mouse Sprn and beta-actin using the primers listed in Table 1. PCR products were verified by gel-electrophoresis. 3. Results 3.1. Generation of the reporter mice Two transgenes were constructed to report on mouse Sprn gene expression. Both contained regulatory sequences of the mouse Sprn gene and either a LacZ or GFP reporter gene (Fig. 1a). These transgenes were microinjected into FVB/N Prnp0/0 mouse eggs [2,16] and reimplanted into surrogate mothers. Genotyping of these mice revealed a normal frequency of transgenic animals born (around 30–35%). All founders transmitted the transgene to their progeny. All the transgenic mouse lines tested, six for Sprn-GFP and three for Sprn-LacZ, expressed the transgene (Fig. 1b).
R. Young et al. / Biochemical and Biophysical Research Communications 412 (2011) 752–756
Expression of the transgene was tested by either fluorescent microscopy (GFP) or LacZ staining (LacZ) of cryosections. The expression of both reporter genes in the cerebellum and thalamus were compared for the six Sprn-GFP and the three Sprn-LacZ lines (Fig. 1c and data not shown). The expression was comparable between the transgenes and between lines carrying the same construct. The expression of Sprn was further analysed on Sprn-LacZ lines (Fig. 2). The reporter mice showed expression of the transgene in the pyramidal cells of the dentate gyrus, in the neurons of the hippocampus, the purkinje cells of the cerebellum, and neurons in the thalamus. For all six Sprn-GFP and three Sprn-LacZ transgenic lines, these results are consistent with the endogenous gene profile, previously described by Watts et al. [1]. 3.2. Gonadic expression of Sprn-LacZ Expression profiles of Sprn-LacZ were investigated in peripheral tissues of the all three transgenic lines using LacZ staining. Tissues collected include thymus, lung, spleen, heart, liver, kidney, intestine, muscle, testicle/ovary, and mammary gland (Supplementary data). RT-PCR analysis was also performed in parallel on the endogenous Sprn gene. RT-PCR and LacZ staining showed tissuespecific expression of endogenous Sprn and the transgene, respectively. LacZ staining showed Sprn-LacZ expression in the male and female gonads (Fig. 2b). In both cases, staining was cell-specific, in the interstitial Leydig cells in the testicle and follicular granulose cells in the ovary. RT-PCR analysis showed the endogenous Sprn gene to be expressed in both of these tissues, however, at a lower level than in the brain (Fig. 2c). 4. Discussion Here we present transgenic reporter mice for the gene encoding Shadoo protein (Sprn). Two transgenes were constructed using the same 5.3 kb regulatory region of the Sprn gene, one using the reporter gene GFP and the other using the reporter gene LacZ. Of the mouse lines established, 100% expressed the GFP or LacZ transgene (6/6 or 3/3 respectively). The expression profile of these transgenes was compared in brain cryosections either by fluorescent microscopy (GFP) or LacZ staining. Both transgenes gave the same expression profile in the regions of the brain analysed (9/9 mouse lines). These expression profiles were compared to the profile of the endogenous gene [1] and were found to be the same, thus validating the profile of these transgenic mice. Further analyses were performed using the Sprn-LacZ mice, various tissues were collected for LacZ staining and mRNA analysis. Of these tissues, brain, testicle and ovary were positive for both the Sprn transgene and endogenous gene. Of the nine other tissues tested, all were negative for both transgene and endogenous gene expression. Sprn-LacZ expression was detected in the Leydig cells of the testis. These cells are located in the interstitial tissue between the seminiferous tubules of the testicles and are the site of testosterone biosynthesis that is required for the development of the male reproductive system, and the initiation and maintenance of spermatogenesis. Testosterone synthesis is dependent on the normal development and differentiation of Leydig cells, however this process is poorly understood. Deregulation of genes expressed in the Leydig cells such as proliferin-related protein (PRP) and mastermind-like domain containing 1 (Mamld1) result in attenuated testosterone production [17,18], and therefore affect the development of the male reproductive system and fertility. Given its expression in the testis, Shadoo could play a role in male fertility. In the mouse ovary, Sprn-LacZ expression was detected in granulosa cells of the ovarian follicles. These follicles follow coordinated development within the ovary, a process tightly regulated
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by hormones and growth factors [19–22]. Both Doppel and PrP expression has been described in female gonads of some species [23,12,24], suggesting prion family proteins may have a role in fertility. The tissue and cell-specific expression of these Sprn-regulated reporter mice allows the study of Sprn expression in adult mice and may serve as a useful tool in deciphering the function of the prion-like protein Shadoo. Furthermore, the described minigene vector could be useful to target any gene expression in Sprn-expressing cells in transgenic mice. Acknowledgments We gratefully thank S. Prusiner for providing the FVB/N Prnp/ mice, Abdelhak Boukadiri for work done with the mice, and Aurélie Auguste for her advice on the staining experiments. RY is a postdoctorant supported by the ANR-09-BLAN-OO15-01. This work was supported by the ANR-09-BLAN-0015-01. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.08.049. References [1] J.C. Watts, B. Drisaldi, V. Ng, J. Yang, B. Strome, P. Horne, M.S. Sy, L. Yoong, R. Young, P. Mastrangelo, C. Bergeron, P.E. Fraser, G.A. Carlson, H.T. Mount, G. Schmitt-Ulms, D. Westaway, The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections, EMBO J. 26 (17) (2007) 4038–4050. [2] H. Büeler, M. Fischer, Y. Lang, H. Bluethmann, H.P. Lipp, S.J. DeArmond, S.B. Prusiner, M. Aguet, C. Weissmann, Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein, Nature 356 (6370) (1992) 577–582. [3] J.C. Manson, A.R. Clarke, M.L. Hooper, L. Aitchison, I. McConnell, J. Hope, 129/ Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal, Mol. Neurobiol. 8 (2–3) (1994) 121–127. [4] J.A. Richt, P. Kasinathan, A.N. Hamir, J. Castilla, T. Sathiyaseelan, F. Vargas, J. Sathiyaseelan, H. Wu, H. Matsushita, J. Koster, S. Kato, I. Ishida, C. Soto, J.M. Robl, Y. Kuroiwa, Production of cattle lacking prion protein, Nat. Biotechnol. 25 (2007) 132–138. [5] G. Yu, J. Chen, Y. Xu, C. Zhu, H. Yu, S. Liu, H. Sha, J. Chen, X. Xu, Y. Wu, A. Zhang, J. Ma, G. Cheng, Generation of goats lacking prion protein, Mol. Reprod. Dev. 76 (2009) 3. [6] V. Zomosa-Signoret, J.D. Arnaud, P. Fontes, M.T. Alvarez-Martinez, J.P. Liautard, Physiological role of the cellular prion protein, Vet. Res. 39 (2008) 9. [7] R. Linden, V.R. Martins, M.A. Prado, M. Cammarota, I. Izquierdo, R.R. Brentani, Physiology of the prion protein, Physiol. Rev. 88 (2008) 673–728. [8] V.R. Martins, F.H. Beraldo, G.N. Hajj, M.H. Lopes, K.S. Lee, M.M. Prado, R. Linden, Prion protein: orchestrating neurotrophic activities, Curr. Issue Mol. Biol. 12 (2009) 63–86. [9] B. Schneider, M. Pietri, E. Pradines, D. Loubet, J.M. Launay, O. Kellermann, S. Mouillet-Richard, Understanding the neurospecificity of prion protein signaling, Front. Biosci. 16 (2011) 169–186. [10] A. Behrens, N. Genoud, H. Naumann, T. Rulicke, F. Janett, F.L. Heppner, B. Ledermann, A. Aguzzi, Absence of the prion protein homologue Doppel causes male sterility, EMBO J. 21 (2002) 3652–3658. [11] D. Paisley, S. Banks, J. Selfridge, N.F. McLennan, A.M. Ritchie, C. McEwan, D.S. Irvine, P.T. Saunders, J.C. Manson, D.W. Melton, Male infertility, DNA damage in Doppel knockout, prion protein/Doppel double-knockout mice, Am. J. Pathol. 164 (2004) 2279–2288. [12] A. Kocer, M. Gallozzi, L. Renault, G. Tilly, I. Pinheiro, F. Le Provost, E. Pailhoux, J.L. Vilotte, Goat PRND expression pattern suggests its involvement in early sex differentiation, Dev. Dyn. 236 (3) (2007) 836–842. [13] M. Premzl, L. Sangiorgio, B. Strumbo, J.A. Marshall Graves, T. Simonic, J.E. Gready, Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein, Gene 314 (2003) 89–102. [14] J.C. Watts, D. Westaway, The prion protein family: diversity rivalry and disfunction, Biochim. Biophys. Acta 1772 (6) (2007) 654–672. [15] R. Young, B. Passet, M. Vilotte, E.P. Cribiu, V. Beringue, F. Le Provost, H. Laude, J.L. Vilotte, The prion or the related Shadoo protein is required for early mouse embryogenesis, FEBS Lett. 583 (2009) 3296–3300. [16] R.K. Giri, R. Young, R. Pitstick, S.J. DeArmond, S.B. Prusiner, G.A. Carlson, Prion infection of mouse neurospheres, Proc. Natl. Acad. Sci. USA 103 (2006) 3875– 3880.
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[17] L. Zhao, J. Hao, J. Hu, Q. Wang, Z. Lü, L. Wang, Q. Yu, Y. Wang, G. Li. Expression of proliferin-related protein in testis and the biological significance in testosterone production. Mol. Cell. Endocrinol. (2011) (EPUB ahead of print). [18] M. Nakamura, M. Fukami, F. Sugawa, M. Miyado, K. Nonomura, T. Ogata, Mamld1 knockdown reduces testosterone production and Cyp17a1 expression in mouse Leydig tumor cells, PLoS One 6 (4) (2011) e19123. [19] A.E. Drummond, M. Dyson, J.E. Mercer, J.K. Findlay, Differential responses of post-natal rat ovarian cells to FSH and activin, Mol. Cell. Endocrinol. 122 (1) (1996) 21–32. [20] E.A. McGee, A.J. Hseuh, Initial cyclic recruitment of ovarian follicles, Endocr. Rev. 21 (2) (2000) 200–214.
[21] J.S. Richards, 2001 Perspective: the ovarian follicle – a perspective in 2001, Endocrinology 142 (2001) 2184–2193. [22] J.E. Fortune, The early stages of follicular development: activation of primordial follicles growth of preantral follicles, Anim. Reprod. Sci. 78 (3-4) (2003) 135–163. [23] M. Rondena, F. Ceciliani, S. Comazzi, V. Pocacqua, C. Bazzocchi, C. Luvoni, S. Chigioni, S. Paltrinieri, Identification of bovine Doppel protein in testis, ovary and ejaculated spermatozoa, Theriogenology 63 (2005) 1195–1206. [24] N. Forde, M. Rogers, M.J. Canty, P. Lonergan, G.W. Smith, P.M. Coussens, J.J. Ireland, A.C. Evans, Association of the prion protein and its expression with ovarian follicle development in cattle, Mol. Reprod. Dev. 75 (2) (2008) 243–249.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice Rachel Young a,⇑, Sandrine Le Guillou a, Gaëlle Tilly a, Bruno Passet a, Marthe Vilotte a, Johan Castille a, Vincent Beringue b, Fabienne Le Provost a, Hubert Laude b, Jean-Luc Vilotte a a b
INRA UMR 1313, Equipe Différenciation et Spécialisation Cellulaires (DISC), Génétique Animale et Biologie Intégrative (GABI), 78350 Jouy-en-Josas, France INRA UR892, Maladies à Prion/Macroassemblage Protéiques, Virologie Immunologie Moléculaires, 78350 Jouy-en-Josas, France
a r t i c l e
i n f o
Article history: Received 8 August 2011 Available online 17 August 2011 Keywords: Transgenic reporter mice Shadoo Paralogue of prion protein Gonadic expression LacZ Fertility and reproduction
a b s t r a c t The protein Shadoo (Sho) is a paralogue of prion protein, and encoded by the gene Sprn. Like prion protein it is primarily expressed in central nervous system, and has been shown to have a similar expression pattern in certain regions of the brain. We have generated reporter mice carrying a transgene encompassing the Sprn promoter, exon 1, intron 1 and the 50 -end of exon 2 driving expression of either the LacZ or GFP reporter gene to study the expression profile of Shadoo in mice. Expression of the reporter genes was analysed in brains of these transgenic mice and was shown to mimic that of the endogenous gene expression, previously described by Watts et al. [1]. Consequently, the Sprn-LacZ mice were used to study the spatial expression of Sho in other tissues of the adult mouse. Several tissues were collected and stained for b-gal activity, including the thymus, heart, lung, liver, kidney, spleen, intestine, muscle, and gonads. From this array of tissues, the transgene was consistently expressed only in specific cell types of the testicle and ovary, suggesting a role for Shadoo in fertility and reproduction. These mice may serve as a useful tool in deciphering the regulation of the prion-like gene Sprn and thus, indirectly, of the Shadoo protein. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The prion protein family consists of three proteins; Prion, Doppel, and Shadoo. Expression profiles of both Prion and Doppel have been characterized in several species including mouse, human, goat, and sheep, however little has been reported on the expression of Sprn. Prion (PrPc) is a ubiquitously expressed protein, with a high expression level in the brain. The function of this GPI-anchored protein is not yet clear, as Prnp-knockout mice [2,3], cattle [4] and goat [5] suffer from no drastic phenotype. An abnormal form of the prion protein, PrPSc, is the infectious agent in prion diseases such as Bovine Spongiform Encephalopathy, Creutzfeldt–Jakob Disease, Scrapie and Chronic Wasting Disease. Various roles have been proposed for PrP in neuroprotection, cellular homeostasis, response to oxidative stress, cell proliferation and differentiation, synaptic function and signal transduction [6–9]. Doppel is highly expressed in the testis, and to a lesser extent the ovaries and spleen. Knockout of the gene encoding Doppel (Prnd) resulted in male sterility [10,11], both studies performed resulted in phenotypes suggesting a role for Doppel in spermatogenesis and/or male fertility. A role for Doppel in early sex differ-
⇑ Corresponding author. Fax: +33 1 34 65 24 78. E-mail address: [email protected] (R. Young). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.049
entiation in goats was also suggested, due to its expression pattern in testis and ovary at various developmental stages [12]. Shadoo expression has been less well characterized than that of Doppel and PrP. The Sprn gene was identified in 2003 by Premzl et al. [13]. Since then homology shared between the N-terminal of the Prion and Shadoo protein structures [14], and overlapping expression pattern in the brain [1] has raised the question of its ability to compensate for lack of PrPc in Prnp-knockout animals [15]. Here we describe transgenic reporter mice generated using Sprn-regulatory sequences and either a green fluorescent protein (GFP) or LacZ reporter gene. These mice were used to analyse the expression profile of Sprn in the adult mouse. 2. Materials and methods All animal manipulations were done according to the ‘‘French Commission de Génie Génétique’’ recommendations. 2.1. Generation of Sprn-GFP and Sprn-LacZ transgenic mice A 5.3 kb genomic fragment including exon 1, intron 1 and the 50 -end of exon 2 of the mouse Sprn gene was amplified using the following primers: sense 50 -CAAGAGGATCTCTATAAACTCAAGGCTA-30 and antisense 50 -AATCTGCGAGAAGAGGGTGGAA-30 . It was
R. Young et al. / Biochemical and Biophysical Research Communications 412 (2011) 752–756
753
Table 1 Sequences of primers used for genotyping and RT-PCR analysis. Gene
Forward primer
Sprn-GFP Sprn-LacZ Endogenous Sprn Beta-actin
Reverse primer
0
0
5 -ACATGAAGCAGCACGACTTCTT-3 50 -GGTCCTAAACCACGCTCCAC-30 50 -CAGTCGTGAGCTCTGCCTAA-30 50 -CACCAGTCCGCCATGGATG-30
50 -TTCTGCTTGTCGGCCATGATATAGACGTT-30 50 -GTTTTCCCAGTCACGACGTTG-30 50 -GGAACAGCTGTCACAGAGGA-30 50 -TCCCCACCATCACACCCTG-30
A
B
GFP
LacZ
Number of mice born
297
208
% Transgenic
35% (6)
33% (3)
% Lines expressing
100% (6/6)
100% (3/3)
C
Fig. 1. Transgene structure, transmission and expression. (A) Two transgenes were made to report on Sprn gene expression in mice, one using a GFP reporter gene and the other using LacZ. In both cases 5.3 kb of the regulatory region of the Sprn gene was used. (B) The table above summarises the mouse lines established for both transgenes. (C) Brain expression profile of Sprn-GFP (green) and Sprn-LacZ (blue). Both transgenes gave the same profile of expression. Cells positive for the transgene resemble the Purkinje cells of the cerebellum and the neurons of the thalamus. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
inserted into either pEGFP-N1 vector or pCMVb vector, to make the Sprn-GFP and Sprn-LacZ constructs respectively. Recombinant vectors were digested with either SalI-AflII (SprnGFP) or SalI (Sprn-LacZ), gel-purified and micro-injected into FVB/N Prnp0/0 oocytes [2,16]. Offspring were genotyped by PCR using the following primers: GFP sense 50 -ACATGAAGCAGCACGACTTCTT-30 , GFP antisense 50 -TTCTGCTTGTCGGCCATGATATAGACGTT-30 , or La cZ sense 50 -GGTCCTAAACCACGCTCCAC-30 , LacZ antisense 50 -GTTT TCCCAGTCACGACGTTG-30 . 2.2. Analysis of transgene expression Three male and three female transgenic mice from each line (six GFP and three LacZ lines) were sacrificed by cervical dislocation.
Three male and three female wild type mice were also used as negative controls. Tissues were collected for either cryosections or RNA extraction in PBS or liquid nitrogen respectively. Sprn-LacZ tissues were fixed in LacZ fix (0.2% gluteraldehyde, 5 mM EGTA, 100 mM MgCl2, PBS) for 4 h then incubated overnight in 20% sucrose solution. Sprn-GFP tissues were incubated in 20% sucrose overnight without fixation. Tissues were mounted in Cryomount (Histolabs) at 25 °C in a cryostat, and then stored at 80 °C. Ten micron cryosections of Sprn-GFP or Sprn-LacZ tissues were cut and collected on Superfrost slides (Thermo Scientific). Sprn-GFP sections were counterstained with DAPI using Vectorshield + DAPI (Vectorlabs). Sprn-LacZ sections were re-fixed for 10 min in LacZ fix, then washed in a detergent wash (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Igepal, PBS) 2 5 min, then
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Fig. 2. Expression of Sprn-LacZ. (A) Brain expression of Sprn-LacZ. Expressing regions include the dentate gyrus, hippocampus, cerebellum and thalamus. The expression pattern given by the transgene mirrors that of the endogenous gene (published by Watts et al. [1]). (B) Sprn-LacZ tissue-distribution. Other tissues were tested, including; testicle, ovary, liver, kidney, lung, thymus, muscle, heart, mammary gland, and intestine. The transgene was only expressed in brain, testicle and ovary. In the testicle, SprnLacZ was expressed in the leydig cells. In the ovary Sprn-LacZ was expressed in the granulosa cells of the developing follicle. Non-transgenic mice were used as negative controls for (A) and (B), these showed no staining (data not shown). (C) RT-PCR analysis of endogenous Sprn correlates with expression of the Sprn-LacZ transgene. Abbreviations: BR, brain; SPL, spleen; TES, testis; INT, intestine; KID, kidney; LV, liver; OV, ovary; THY, thymus; HRT, heart.
stained overnight at 37 °C in a humid staining chamber with LacZ stain (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Igepal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 20 mM Tris buffer pH 7.3, 1 mg/ml X-gal, PBS) protected from the light. The sections are then washed 3 5 min in PBS then rinsed in dH2O, air dried and mounted with Permount (Fischer Scientific). Sprn-GFP sections were analysed by fluorescent light microscopy using a Leica microscope. Sprn-LacZ sections were analysed by light microscopy on the same microscope. 2.3. RNA extractions and RT-PCR RNA collected for RT-PCR analysis was extracted using the RNeasy Lipid Tissue Mini kit (Qiagen). RNA concentration was calculated by electro-spectrophotometry, then 5 lg of each sample was reverse transcribed using the Superscript III Reverse Transcription kit (Invitrogen).
RT-PCR analysis was performed for both endogenous mouse Sprn and beta-actin using the primers listed in Table 1. PCR products were verified by gel-electrophoresis. 3. Results 3.1. Generation of the reporter mice Two transgenes were constructed to report on mouse Sprn gene expression. Both contained regulatory sequences of the mouse Sprn gene and either a LacZ or GFP reporter gene (Fig. 1a). These transgenes were microinjected into FVB/N Prnp0/0 mouse eggs [2,16] and reimplanted into surrogate mothers. Genotyping of these mice revealed a normal frequency of transgenic animals born (around 30–35%). All founders transmitted the transgene to their progeny. All the transgenic mouse lines tested, six for Sprn-GFP and three for Sprn-LacZ, expressed the transgene (Fig. 1b).
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Expression of the transgene was tested by either fluorescent microscopy (GFP) or LacZ staining (LacZ) of cryosections. The expression of both reporter genes in the cerebellum and thalamus were compared for the six Sprn-GFP and the three Sprn-LacZ lines (Fig. 1c and data not shown). The expression was comparable between the transgenes and between lines carrying the same construct. The expression of Sprn was further analysed on Sprn-LacZ lines (Fig. 2). The reporter mice showed expression of the transgene in the pyramidal cells of the dentate gyrus, in the neurons of the hippocampus, the purkinje cells of the cerebellum, and neurons in the thalamus. For all six Sprn-GFP and three Sprn-LacZ transgenic lines, these results are consistent with the endogenous gene profile, previously described by Watts et al. [1]. 3.2. Gonadic expression of Sprn-LacZ Expression profiles of Sprn-LacZ were investigated in peripheral tissues of the all three transgenic lines using LacZ staining. Tissues collected include thymus, lung, spleen, heart, liver, kidney, intestine, muscle, testicle/ovary, and mammary gland (Supplementary data). RT-PCR analysis was also performed in parallel on the endogenous Sprn gene. RT-PCR and LacZ staining showed tissuespecific expression of endogenous Sprn and the transgene, respectively. LacZ staining showed Sprn-LacZ expression in the male and female gonads (Fig. 2b). In both cases, staining was cell-specific, in the interstitial Leydig cells in the testicle and follicular granulose cells in the ovary. RT-PCR analysis showed the endogenous Sprn gene to be expressed in both of these tissues, however, at a lower level than in the brain (Fig. 2c). 4. Discussion Here we present transgenic reporter mice for the gene encoding Shadoo protein (Sprn). Two transgenes were constructed using the same 5.3 kb regulatory region of the Sprn gene, one using the reporter gene GFP and the other using the reporter gene LacZ. Of the mouse lines established, 100% expressed the GFP or LacZ transgene (6/6 or 3/3 respectively). The expression profile of these transgenes was compared in brain cryosections either by fluorescent microscopy (GFP) or LacZ staining. Both transgenes gave the same expression profile in the regions of the brain analysed (9/9 mouse lines). These expression profiles were compared to the profile of the endogenous gene [1] and were found to be the same, thus validating the profile of these transgenic mice. Further analyses were performed using the Sprn-LacZ mice, various tissues were collected for LacZ staining and mRNA analysis. Of these tissues, brain, testicle and ovary were positive for both the Sprn transgene and endogenous gene. Of the nine other tissues tested, all were negative for both transgene and endogenous gene expression. Sprn-LacZ expression was detected in the Leydig cells of the testis. These cells are located in the interstitial tissue between the seminiferous tubules of the testicles and are the site of testosterone biosynthesis that is required for the development of the male reproductive system, and the initiation and maintenance of spermatogenesis. Testosterone synthesis is dependent on the normal development and differentiation of Leydig cells, however this process is poorly understood. Deregulation of genes expressed in the Leydig cells such as proliferin-related protein (PRP) and mastermind-like domain containing 1 (Mamld1) result in attenuated testosterone production [17,18], and therefore affect the development of the male reproductive system and fertility. Given its expression in the testis, Shadoo could play a role in male fertility. In the mouse ovary, Sprn-LacZ expression was detected in granulosa cells of the ovarian follicles. These follicles follow coordinated development within the ovary, a process tightly regulated
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by hormones and growth factors [19–22]. Both Doppel and PrP expression has been described in female gonads of some species [23,12,24], suggesting prion family proteins may have a role in fertility. The tissue and cell-specific expression of these Sprn-regulated reporter mice allows the study of Sprn expression in adult mice and may serve as a useful tool in deciphering the function of the prion-like protein Shadoo. Furthermore, the described minigene vector could be useful to target any gene expression in Sprn-expressing cells in transgenic mice. Acknowledgments We gratefully thank S. Prusiner for providing the FVB/N Prnp/ mice, Abdelhak Boukadiri for work done with the mice, and Aurélie Auguste for her advice on the staining experiments. RY is a postdoctorant supported by the ANR-09-BLAN-OO15-01. This work was supported by the ANR-09-BLAN-0015-01. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.08.049. References [1] J.C. Watts, B. Drisaldi, V. Ng, J. Yang, B. Strome, P. Horne, M.S. Sy, L. Yoong, R. Young, P. Mastrangelo, C. Bergeron, P.E. Fraser, G.A. Carlson, H.T. Mount, G. Schmitt-Ulms, D. Westaway, The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections, EMBO J. 26 (17) (2007) 4038–4050. [2] H. Büeler, M. Fischer, Y. Lang, H. Bluethmann, H.P. Lipp, S.J. DeArmond, S.B. Prusiner, M. Aguet, C. Weissmann, Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein, Nature 356 (6370) (1992) 577–582. [3] J.C. Manson, A.R. Clarke, M.L. Hooper, L. Aitchison, I. McConnell, J. Hope, 129/ Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal, Mol. Neurobiol. 8 (2–3) (1994) 121–127. [4] J.A. Richt, P. Kasinathan, A.N. Hamir, J. Castilla, T. Sathiyaseelan, F. Vargas, J. Sathiyaseelan, H. Wu, H. Matsushita, J. Koster, S. Kato, I. Ishida, C. Soto, J.M. Robl, Y. Kuroiwa, Production of cattle lacking prion protein, Nat. Biotechnol. 25 (2007) 132–138. [5] G. Yu, J. Chen, Y. Xu, C. Zhu, H. Yu, S. Liu, H. Sha, J. Chen, X. Xu, Y. Wu, A. Zhang, J. Ma, G. Cheng, Generation of goats lacking prion protein, Mol. Reprod. Dev. 76 (2009) 3. [6] V. Zomosa-Signoret, J.D. Arnaud, P. Fontes, M.T. Alvarez-Martinez, J.P. Liautard, Physiological role of the cellular prion protein, Vet. Res. 39 (2008) 9. [7] R. Linden, V.R. Martins, M.A. Prado, M. Cammarota, I. Izquierdo, R.R. Brentani, Physiology of the prion protein, Physiol. Rev. 88 (2008) 673–728. [8] V.R. Martins, F.H. Beraldo, G.N. Hajj, M.H. Lopes, K.S. Lee, M.M. Prado, R. Linden, Prion protein: orchestrating neurotrophic activities, Curr. Issue Mol. Biol. 12 (2009) 63–86. [9] B. Schneider, M. Pietri, E. Pradines, D. Loubet, J.M. Launay, O. Kellermann, S. Mouillet-Richard, Understanding the neurospecificity of prion protein signaling, Front. Biosci. 16 (2011) 169–186. [10] A. Behrens, N. Genoud, H. Naumann, T. Rulicke, F. Janett, F.L. Heppner, B. Ledermann, A. Aguzzi, Absence of the prion protein homologue Doppel causes male sterility, EMBO J. 21 (2002) 3652–3658. [11] D. Paisley, S. Banks, J. Selfridge, N.F. McLennan, A.M. Ritchie, C. McEwan, D.S. Irvine, P.T. Saunders, J.C. Manson, D.W. Melton, Male infertility, DNA damage in Doppel knockout, prion protein/Doppel double-knockout mice, Am. J. Pathol. 164 (2004) 2279–2288. [12] A. Kocer, M. Gallozzi, L. Renault, G. Tilly, I. Pinheiro, F. Le Provost, E. Pailhoux, J.L. Vilotte, Goat PRND expression pattern suggests its involvement in early sex differentiation, Dev. Dyn. 236 (3) (2007) 836–842. [13] M. Premzl, L. Sangiorgio, B. Strumbo, J.A. Marshall Graves, T. Simonic, J.E. Gready, Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein, Gene 314 (2003) 89–102. [14] J.C. Watts, D. Westaway, The prion protein family: diversity rivalry and disfunction, Biochim. Biophys. Acta 1772 (6) (2007) 654–672. [15] R. Young, B. Passet, M. Vilotte, E.P. Cribiu, V. Beringue, F. Le Provost, H. Laude, J.L. Vilotte, The prion or the related Shadoo protein is required for early mouse embryogenesis, FEBS Lett. 583 (2009) 3296–3300. [16] R.K. Giri, R. Young, R. Pitstick, S.J. DeArmond, S.B. Prusiner, G.A. Carlson, Prion infection of mouse neurospheres, Proc. Natl. Acad. Sci. USA 103 (2006) 3875– 3880.
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